Recent progress in our understanding of the molecular biology of estrogen receptor action has provided a great deal of evidence which promises to increase our understanding of the mechanism through which SERMs elicit their tissue-specific effects. This in turn has enhanced interest in raloxifene and increased the interest in developing new tissue-specific SERMs. The identification of numerous coactivators and corepressors150,151 which modulate receptor function and the realization that there are two subtypes of estrogen receptor (ERa and ERb)152 attest to the potential complexity through which SERMs produce diverse tissue-specific responses. To date, more than 20 coregulator proteins have been discovered that bind to estrogen receptors and modulate their function, each acting as either a positive (coactivator) or a negative (corepressor) transcriptional regulator. Depending on the unique receptor conformation induced by ligand binding, varying combinations of coregulator proteins can potentially interact with the estrogen receptor complex to modulate its function in a variety of ways.150,151
What separates SERMs like raloxifene and tamoxifen from classical estrogen agonists (i.e., E2) is their ability to function in a similar fashion to the classical estrogens on some tissues while not acting as agonists, or perhaps even behaving as antagonists in other tissues. During the late 1990s there was an increased interest in the molecular mechanism of action of raloxifene by the scientific community. This in turn enhanced interest in the development of raloxifene as a clinically useful agent. There was also interest in determining the mechanism of drug resistance to tamoxifen. Development of tamoxifen resistance can be characterized by an increase in the partial agonist properties of the antiestrogen in the breast, resulting in loss of growth inhibition and even inappropriate tumor stimulation. Initially, it was thought that a mutation in the estrogen receptor might be involved in changing the pharmacology of tamoxifen from an antiestrogen to an estrogen; however, no such mutations were ever found in clinical samples. Interestingly, the chance finding of a D351Y (Asp-351 to Tyr-351) estrogen receptor mutation in a tamoxifen-stimulated breast tumor in 1994, ' that enhanced the agonist activity of 4-hydroxytamoxifen and altered the pharmacology of raloxifene from an antiestrogen to a partial agonist,155,156 provided an invaluable starting point to decipher how SERMs modulate estrogen receptor function.
Evidence from co-crystal structures of estrogen receptor ligand-binding domains complexed with an estrogen or a SERM (i.e., tamoxifen or raloxifene) provided important information as to how an estrogen or antiestrogen alters the shape of the estrogen receptor complex.157,158 A key feature of the estrogen agonist-ERa structure is the ability of the ligand to be enveloped in a hydrophobic pocket that is closed by helix 12 in the ligand-binding domain of ERa. This change in structure (i.e., repositioning of helix 12) facilitates coactivator binding to the AF-2 region of the receptor and is considered an important mechanism for full estrogen action at ERa.157 Both raloxifene and 4-hydroxytamoxifen fit into the hydrophobic pocket of the ligand-binding domain; however, the bulky alkylaminoethoxyphenyl side chain prevents the reorientation of helix 12 that must seal the ligand into the receptor before coactivators can bind and produce a transcription complex. The high-affinity antiestrogens both interact through phenolic hydroxyls with Glu-353 and Arg-394 to locate the ligands correctly in the binding domain157; however, the side chain, which is critical for antiestrogenic activity, interacts with an aspartic acid residue (Asp-351; D351), which lies at the base of ERa helix 3.157,158 An examination of the surface structure of the raloxifene-estrogen receptor complex has shown that Asp-351 forms a strong hydrogen bond (2.7A, 180°) with the tertiary amine of the piperidine ring of the antiestrogenic side chain of raloxifene.157 This interaction forces the piperidine ring into an awkward high-energy gauche position so that the bulky side chain of raloxifene can shield and neutralize the Asp-351 (D351) on the receptor surface.159 It is suggested that the shielding or neutralization of D351 by the side chain of raloxifene is responsible for the difference in the intrinsic activity of the raloxifene and tamoxifen-ERa complex. Replacing the aspartate with glycine results in a tamoxifen D351G-ERa complex that has lost estrogen-like activity while retaining antiestrogenic properties.160,161 The D351G mutation decreases the affinity of raloxifene for ERa, thereby illustrating the important role of the interaction of the piperidine side chain and D351. Similarly, the raloxifene-ERa complex can be modulated through both D351 and the antiestrogenic side chain. A D351E mutation that extends the interactive distance from 2.7 A in raloxifene D351 to 3.5-5 A in E351 increases the estrogen-like action of the raloxifene-ERa complex.159
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